Collagen scaffolds

ABSTRACT

Methods for preparing and using collagen extracts and collagen scaffolds are provided. Additionally methods and related kits for the repair of articular tissue using the collagen material are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/679,629, filed Aug. 17, 2017, which is continuation of U.S.application Ser. No. 14/765,064, filed Jul. 31, 2015, which is nationalstage entry under 35 U.S.C. 371 of PCT Application No.PCT/US2014/014141, filed Jan. 31, 2014, which claims priority under 35U.S.C. § 119(e) to U.S. provisional patent application, U.S. Ser. No.61/759,868, filed Feb. 1, 2013, and entitled Collagen Scaffolds. Theentire contents of each application listed in this paragraph areincorporated herein by reference.

BACKGROUND OF THE INVENTION

While the body has efficient processes for healing most damaged tissue,tissue such as intra-articular tissue often fails to heal after aninjury. The tissue outside of joints heals by forming a fibrin clot,which connects the ruptured tissue ends and is subsequently remodeled toform scar, which heals the tissue. Inside a synovial joint, a fibrinclot either fails to form or is quickly lysed after injury to the knee,thus preventing joint arthrosis and stiffness after minor injury. Jointscontain synovial fluid which, as part of normal joint activity,naturally prevent clot formation in joints. This fibrinolytic processresults in premature loss of the fibrin clot scaffold and disruption ofthe healing process for tissues within the joint or withinintra-articular tissues. Enhancing healing of ligaments using growthfactors has been an area of great interest and research.

SUMMARY OF THE INVENTION

The invention relates in some aspects to methods for preparing and usingcollagen extracts and scaffolds and related products.

In some aspects the invention is a method for preparing a collagenextract. The method involves one or more of the steps of animal tissuedissection, freeze drying, such as lyophilization, salt extraction,water rinse and detergent treatment, PBS rinse and wash, enzymedigestion, PBS/EDTA wash, water rinse and citrate buffer, andultracentrifugation and acid solubilization.

Once the collagen extract is prepared the collagen scaffold can be madeusing the collagen extract.

In some embodiments, the animal tissue dissection involves retrieval oftissues from porcine, ovine, bovine or human knees. In some embodiments,the animal tissue dissection involves retrieval of tissue either fromfresh or previously frozen (and then thawed) knees (from age specificanimals with skin on to maintain sterility). In other embodiments theknee connective tissue is dissected from fresh and skin-on knees understerile conditions. If the dissection is from fresh knees the tissue maybe frozen before the first step. In some embodiments, the tissueharvested is dermis. In some embodiments, the tissue harvested issubdermal tissue. In some embodiments, the tissue harvested is adiposetissue. In some embodiments, the tissue harvested is bursal tissue. Insome embodiments, the tissue harvested is joint capsule. In someembodiments, the tissue harvested is ligament. In some embodiments, thetissue harvested is tendon. In some embodiments, the tissue harvested iscartilage. In some embodiments, the tissue harvested is meniscus. Insome embodiments, the tissue harvested is muscle. In some embodiments,the tissue harvested is subdermal muscle fascia.

In some embodiments, the freeze drying step involves putting the samplesinto the 4° C. refrigerator for at least 3 hours followed bylyophilization. In other embodiments, the freeze drying step involvesputting the samples into the −20° C. freezer for at least 3 hoursfollowed by lyophilization. In other embodiments the freeze drying stepinvolves putting the samples into the −80° C. freezer for at least 3hours followed by lyophilization. In other embodiments the time in therefrigerator or freezer is as short as 30 minutes or as long as oneyear.

In some embodiments, the homogenization step involves retrieving tissuefrom Petri dishes using a sterile forceps and putting them in specifiedvolumes into a homogenizer, blender or food processor with some crusheddry ice. In other embodiments, the homogenizing vessel is kept cold byexternal cooling with dry ice or a circulating bath. In otherembodiments, the homogenizing vessel is placed into a cold room for thisstep. The homogenizer may be run for about 2 minutes until the bigpieces of source tissue have disappeared. The homogenizer may be run forlonger periods as well. The material may then be placed into pre-weighedempty 50 ml centrifuge tubes, filling the tube up to 20 ml line. Theprocessed tissue may be stored in a sealed container at 4° C. if notused immediately and the powdered tissue can be stored at 4° C. for amonth. In other embodiments, the tissue may be stored at −20 degrees C.for up to one year. In other embodiments, the tissue may be stored at−80 degrees C. for up to three years. Once the tissue weight isdetermined it can be recorded for later use. Each sample may then befilled up to the 45 ml line with 2% antibiotic-antimycotic in 1XPB S androcked at 40° C. In other embodiments, it may be placed into anantibiotic solution at 4 degrees C. Once the tissue is pelleted theantibiotic-antimycotic solution may be aspirated into a container with10% low mercury bleach and the pellet of tissue placed on ice.

In some embodiments the water rinse and detergent treatment involves thesteps rinsing each sample three times with sterile ultrapure (Milli-Q)water. After the last supernatant is removed, the tubes may be filled upto the 45 ml line with sterile PBS with a detergent solution and placedon a rocker at 37° C. for 24 h. In other embodiments, this step mayoccur at a different temperature or without agitation.

In other embodiments the PBS rinse and wash involves spinning eachsample down at 3,000 rpm for 5 minutes to “pellet” the samples, followedby a rinse with sterile PBS. In some embodiments the method may alsoinvolve filling the tubes to 45 ml line with PBS, and placing the tubeson a rocker at 4° C. for 24 h. In other embodiments, this step may occurat a different temperature or without agitation.

The first enzyme digestion in some embodiments involves, a rinse of thepelleted material with sterile PBS and then filling each tube up to the45 ml line with Enzyme solution A, and placing the tube on a rocker at37° C. for 24 h. In other embodiments, the tissue is in contact withEnzyme A for time periods up to 2 weeks. In other embodiments, thetissue and Enzyme A are placed on a rocker at a different temperature.

The PBS/EDTA wash in some embodiments involves rinsing each sample oncewith sterile PBS followed by incubation with PBS containing EDTA (eachtube filled up to the 45 ml line) for 24 hr at 4° C. In otherembodiments, this step may occur at a different temperature or withagitation.

In some embodiments the second extraction involves incubation of thepellet with sterile CaCl extraction solution. In other embodiments, thesecond extraction involves incubation of the pellet with a differentsterile salt extraction solution (NaCl, KCl or similar) in the cold room(at 4° C.) for 24 h. In other embodiments, the second extractioninvolves incubation of the pellet with a sterile salt extractionsolution (each tube filled to the 45 ml line) at room temperature.

In other embodiments the water rinse and citrate buffer step involvesrinsing each pelleted sample three times with 25-30 ml of sterileultrapure (Milli-Q) water at room temperature for 15 min followed bymixing the pellet obtained by centrifugation with sterile citrate buffer(tubes filled to 45 ml line) at 4° C. for 72 h.

The ultracentrifugation and acid solubilization step, in someembodiments involves ultra-centrifugation to obtain a pellet, pullingthe pellet apart before transferring into a endotoxin-free 1 or 2 literglass bottle and adding approximately 80% of the estimated final volumeof sterile acid (HCl, HAc or similar). The bottle may be agitated toloosen up the pellet and then placed at 4° C. for 48 h. In otherembodiments, a different acid solution may be used. In otherembodiments, the acid solution and pellet are kept together for timepoints up to one year, and they may be kept at −20 or −80 degrees C.

In some embodiments the pepsin digestion is performed by adding apepsin/acid solution into the sample and rocked until homogeneity of theslurry is achieved to produce the collagen extract.

The collagen scaffold may be prepared from the collagen extract. In someembodiments the collagen extract is lyophilized until completely dry. Inother embodiments, only a fraction of the water is removed from theslurry to concentrate it. In other embodiments the lyophilized collagenslurry can be reconstituted in ultrapure water at a desiredconcentration and centrifuged. In yet other embodiments, a buffersolution is added and the mixtures is put on rocker until mixed, andthen calcium is added and mixed within the tube. In some embodiments,the a calcium solution is made before the day of use and stored at −20degrees C. In other embodiments, the calcium solution is made on the dayof use and stored at room temperature. In other embodiments, a calciumcarbonate, calcium chloride or calcium gluconate solution is used. Thecollagen slurry may be neutralized with the addition of KOH, NaOH oranother acid or base. In yet other embodiments the collagen slurry maybe added to a mold and centrifuged (3500 rpm for 5 min at 4 C) to removeair bubbles, and then placed in a warm environment to gel. In otherembodiments, the slurry is frozen after addition of the acid or base. Inother embodiments, the slurry is maintained at room temperature untilthe viscosity increases. In other embodiments the collagen is thenfrozen and lyophilized to produce a collagen scaffold. The collagenscaffold may be used in any of the methods described herein.

In some aspects, the invention is a method for preparing a collagenextract by freezing a source tissue; preparing ground tissue by cuttingthe source tissue into small pieces using a homogenizer or similardevice while keeping the tissue cold, performing a salt extraction onthe ground tissue to produce a salt extracted collagen, and performing apepsin digestion of the salt extracted collagen to produce a collagenextract.

According to some embodiments the method further involves neutralizingthe collagen extract to produce a neutralized collagen slurry andsubjecting the neutralized collagen slurry to an elevated temperaturesuch that the collagen forms a collagen scaffold comprised ofself-assembled collagen fibers.

In some embodiments the freeze dried collagenous tissue is ground usinga homogenizer. In other embodiments the freeze dried collagenous tissueis ground using a blender with the blender components kept chilledduring processing. The freeze dried collagenous tissue may be cut intosmall pieces with a knife or blade, for example. Alternatively thefreeze dried collagenous tissue is ground using a food processor withthe bowl of the processor kept on ice.

In some embodiments the collagen extract is treated with enzymes orother chemicals to remove cellular debris. In other embodiments thecollagen extract is treated with hyperosmotic solutions to removecellular debris. In yet other embodiments the collagen extract istreated with detergents to remove cellular debris.

In some aspects, the invention is a method of making a collagen scaffoldby preparing a neutralized collagen slurry by mixing a collagen slurrywith a buffer and then with a calcium-containing solution, heating theneutralized collagen slurry, and freeze drying the heated neutralizedcollagen slurry to produce the collagen scaffold. Optionally a step offreeze drying the collagen extract is performed prior to theneutralization step.

In another aspect, the invention is a collagen extract or collagenscaffold preparable by any of the methods described herein.

In other aspects a kit, including a first container housing asolubilized collagen solution prepared according to the methodsdescribed herein, a buffer housed in the first container or in a secondcontainer, optionally a calcium-containing solution housed in the firstor second containers or in a third container and instructions forpreparing a collagen scaffold is provided.

The kit may also include a container housing a neutralization solution.

The invention according to other aspects is a method comprisingcontacting the ends of a ruptured articular tissue in a subject with asolubilized collagen scaffold prepared as described herein, and allowingthe solution to set to treat the ruptured tissue.

In some embodiments the articular tissue is intra-articular tissue. Anintra-articular injury may be, for instance, a meniscal tear, ligamenttear or a cartilage lesion.

In other embodiments the articular tissue is extra-articular tissue. Anextra-articular injury may be, for instance, ligament, tendon or muscleinjury.

The method may involve mechanically joining the ends of the rupturedtissue.

Any of the collagen materials described herein can be substantially freeof one or more of the following: nucleic acid (DNA and/or RNA),glycosaminoglycan (GAG), phospholipid, active pepsin, and active virus.In other embodiments, the collagen material can have a substantiallyreduced level of one or more of the following: nucleic acid (DNA and/orRNA), glycosaminoglycan (GAG), phospholipid, active pepsin, and activevirus. In one example, the content of phospholipid in the collagenmaterial is less than 20% (e.g., less than 15%, 10%, 5%, or 1%) of thatfound in a native tissue. The content of phospholipid in the collagenmaterial can be less than 10,000 μM/mg, 5,000 μM/mg, 2,500 μM/mg, 1,250μM/mg, 1,000 μM/mg, 500 μM/mg, 125 μM/mg, or 50 μM/mg. In anotherexample, the content of nucleic acids (e.g., DNA or RNA) in the collagenmaterial is less than 20% (e.g., less than 15%, 10%, 5%, or 1%) of thatfound in a native tissue. The content of nucleic acids in the collagenmaterial can be less than 700 μg/g, 350 μg/g, 200 μg/g, 100 μg/g, 35μg/g, 10 μg/g, 5 μg/g, 1 μg/g, 0.5 μg/g, or 0.25 μg/g. In yet anotherexample, the level of active pepsin in the material is less than 10,000μg/ml (e.g., 1,000 μg/ml or 200 μg/ml). In still another example, thecontent of GAG in the collagen material is less than 50% of the totalmaterial (e.g., less than 40%, 30%, 20%, 10%, or 5%).

Any of the collagen materials described herein can be treated byterminal sterilization, e.g., ethylene oxide sterilization or electronbeam sterilization. In some embodiments, the ethylene oxidesterilization may be conducted under specific conditions (e.g., thosedescribed in Examples below). For example, the ethylene oxidesterilization may be conducted with a cycle temperature of less than 120degrees F., e.g., less than 110 degrees F., 100 degrees F., or 90degrees F. Prior to the sterilization, the collagen material can belyophilized. Alternatively or in addition, the collagen material can berehydrated after the sterilization.

In another aspect, described herein is an extracellular matrix (ECM)material (e.g., an ECM scaffold such as a collagen scaffold), comprisingat least one extracellular matrix component (e.g., collagen or anon-collagen ECM component), calcium, and optionally a platelet, whereinthe content of calcium in the ECM solution for preparing the ECMscaffold ranges from 1-5 mg/g (calcium/ECM solution) or about 0.005-10 gCaCl₂ per gram of the ECM component (e.g., collagen), for example, 1-5mg CaCl₂/40 mg ECM protein (e.g., collagen). In another embodiment, thecalcium content can range from 1 to 5 gm CaCl₂ to each gram of collagenin the biomaterial as described herein. In another embodiment, 10 to 200mM of calcium can be added to the collagen material or scaffold. Such anECM scaffold can further comprise one or more of the following: growthfactor, platelet, white blood cell, stem cell, cross-linker, andneutralizing agent. In some examples, the ECM scaffold is prepared froman ECM solution comprising at least 100 mOsm calcium per kilogram ofcollagen solution. In other examples, the ECM scaffold is prepared froman ECM solution comprising at least 90 mOsm (e.g., 80, 70, 60, 50, 40,30, 20, or 10 mOsm) calcium per liter.

Any of the calcium-containing biomaterials, such as collagen materials,as described herein, can be substantially free of one or more of thefollowing: nucleic acid (DNA and/or RNA), glycosaminoglycan (GAG),phospholipid, active pepsin, and active virus. In other embodiments, thecollagen material can have a substantially reduced level of one or moreof the following: nucleic acid (DNA and/or RNA), glycosaminoglycan(GAG), phospholipid, active pepsin, and active virus. In one example,the content of phospholipid in the collagen material is less than 20%(e.g., less than 15%, 10%, 5%, or 1%) of that found in a native tissue.The content of phospholipid in the collagen material can be less than10,000 μM/mg, 5,000 μM/mg, 2,500 μM/mg, 1,250 μM/mg, 1,000 μM/mg, 500μM/mg, 125 μM/mg, or 50 μM/mg. In another example, the content ofnucleic acids (e.g., DNA or RNA) in the collagen material is less than20% (e.g., less than 15%, 10%, 5%, or 1%) of that found in a nativetissue. The content of nucleic acids in the collagen material can beless than 700 μg/g, 350 μg/g, 200 μg/g, 100 μg/g, 35 μg/g, 10 μg/g, 5μg/g, 1 μg/g, 0.5 μg/g, or 0.25 μg/g. In yet another example, the levelof active pepsin in the material is less than 10,000 μg/ml (e.g., 1,000μg/ml or 200 μg/ml). In still another example, the content of GAG in thecollagen material is less than 50% of the total material (e.g., lessthan 40%, 30%, 20%, 10%, or 5%).

In some embodiments, the biomaterial described herein (e.g., a collagenmaterial) comprises GAG. The content of GAG in such a biomaterial can beat least 20% of the total dry weight of the biomaterial, for example, atleast 30%, 40%, or 50% of the total dry weight of the biomaterial. Insome examples, the GAG-containing biomaterial is substantial free ofnucleic acids (e.g., DNA and/or RNA), phospholipid, active pepsin,and/or active virus as described herein. In one example, the content ofphospholipid in the collagen material is less than 20% (e.g., less than15%, 10%, 5%, or 1%) of that found in a native tissue. The content ofphospholipid in the collagen material can be less than 10,000 μM/mg,5,000 μM/mg, 2,500 μM/mg, 1,250 μM/mg, 1,000 μM/mg, 500 μM/mg, 125μM/mg, or 50 μM/mg. In another example, the content of nucleic acids(e.g., DNA or RNA) in the collagen material is less than 20% (e.g., lessthan 15%, 10%, 5%, or 1%) of that found in a native tissue. The contentof nucleic acids in the collagen material can be less than 700 μg/g, 350μg/g, 200 μg/g, 100 μg/g, 35 μg/g, 10 μg/g, 5 μg/g, 1 μg/g, 0.5 μg/g, or0.25 μg/g. In yet another example, the level of active pepsin in thematerial is less than 10,000 μg/ml (e.g., 1,000 μg/ml or 200 μg/ml).

In another aspect, the present disclosure provides a method forpreparing extracellular matrix (ECM) scaffolds such as collagenscaffolds that comprise calcium. Such calcium-containing scaffolds arealso described herein.

In some examples, the method for preparing the calcium-containing ECMscaffold comprises: mixing a composition comprising at least one ECMcomponent (e.g., those described herein such as collagen) with a calciumsolution to form a mixture; lyophilizing the mixture to form an ECMsponge (e.g., a collagen sponge); and neutralizing the ECM sponge (e.g.,via a HEPES buffer) to produce the calcium-containing ECM scaffold.Optionally, the ECM sponge can be rehydrated prior to theneutralization.

In other examples, the method for preparing the calcium-containing ECMscaffold comprises: soaking an ECM sponge in a calcium solution, whereinthe ECM sponge comprises at least one ECM component (e.g., thosedescribed herein such as collagen) and is neutralized; and lyophilizingthe ECM sponge to form the calcium-containing ECM scaffold. The methodcan further comprise the following steps for preparing the ECM sponge(e.g., collagen sponge): neutralizing a slurry containing at least oneECM component such as collagen to form a neutralized ECM slurry (e.g.,collagen slurry); incubating the neutralized ECM slurry to allowgelation of the slurry; and lyophilizing the ECM material (e.g.,collagen material) thus formed to produce the ECM sponge. Prior to theneutralizing step, the method can further comprise: lyophilizing an ECMsolution (e.g., a collagen solution); and rehydrating the lyophilizedECM solution to form the ECM slurry.

In any of the methods for preparing the calcium-containing ECM scaffolddescribed herein, the calcium solution can have a calcium concentrationof about 30 mM to 90 mM. Alternatively or in addition, the ratio ofcalcium to collagen is about (0.005-10):1. Each of the limitations ofthe invention can encompass various embodiments of the invention. It is,therefore, anticipated that each of the limitations of the inventioninvolving any one element or combinations of elements can be included ineach aspect of the invention. This invention is not limited in itsapplication to the details of construction and the arrangement ofcomponents set forth in the following description or illustrated in thedrawings. The invention is capable of other embodiments and of beingpracticed or of being carried out in various ways. Also, the phraseologyand terminology used herein is for the purpose of description and shouldnot be regarded as limiting. The use of “including”, “comprising”, or“having”, “containing”, “involving”, and variations thereof herein, ismeant to encompass the items listed thereafter and equivalents thereofas well as additional items.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph showing initial packaging set up of an ECMScaffold (e.g., a collagen scaffold) for e-beam testing.

FIG. 2 is a chart showing DNA content in samples treated as described inExample 2 below.

FIGS. 3A-3C depict DNA content (3A), GAG content (3B), and phospholipidcontent (3C) in samples treated as described in Example 2 below relativeto the DNA, GAG, and phospholipid content in native tissues and incommercially available collagen scaffold.

FIG. 4 is a chart showing the level of active pepsin in collagenmaterials treated as described in Example 3 below.

DETAILED DESCRIPTION OF THE INVENTION

The invention involves, in some aspects, methods for preparing collagenextracts. A detailed protocol for the method is provided below. Thecollagen extracts prepared according to the methods have superiorproperties to collagen extracts prepared according to other methods andare particularly useful as a collagen source for preparing tissuescaffolds. The methods involve a number of steps, many of which areperformed in other extraction procedures. However, when the steps werecompiled as described in the various aspects and embodiments presentedin the specification the quality of the product produced by those stepswas unexpected.

A detailed protocol is provided below. Cadaveric knees from the desiredspecies are obtained. If obtained frozen, they are thawed beforeharvesting the desired tissues. The knees are prepared with solutions toreduce the bacterial load on the skin and then the skin is incised andthe underlying layers split until the desired tissue is reached. Thetissue is harvested and placed into sterile containers. At this point,the tissue may be frozen at −20 or −80 degrees C. and stored for up toone year. Alternatively, the tissue can by lyophilized immediately.

After lyophilization, the tissue is homogenized using a blender,homogenizer, food processor, scalpel or some combination of thesedevices. An alternate device for cutting tissue can also be used. Thesmall pieces of tissue are placed into a sterile salt solution which maycontain antibiotics and/or an antimycotic. In one embodiment, thehomogenization may be carried out at room temperature. In anotherembodiment, the homogenization may be carried out below roomtemperature. In another embodiment, the homogenization may be carriedout at 4 degrees C. In another embodiment, the homogenization may becarried out below 4 degrees C. In one embodiment, dry ice is used tomaintain the desired temperature of the tissue. In another embodiment,the homogenizing instrument is cooled to maintain the desiredtemperature. In another embodiment, the homogenizing vessel is cooled tomaintain the desired temperature. In another embodiment, all parts ofthe homogenizing elements are sterilizable. In another embodiment, allinstrumentation which could potentially contact the tissue issterilizable. In another embodiment, all instrumentation which couldpotentially contact the tissue is sterile. In another embodiment, allinstrumentation which could potentially contact the tissue issubstantially free of endotoxin.

A salt extraction may be used to treat the tissue. The homogenizedtissue may be placed into a tube containing a salt solution at a givenconcentration. In one embodiment, the solution is a 10% salt solution.In another embodiment, the solution is a 20% salt solution. In anotherembodiment, the solution is a 30% salt solution. In another embodiment,the solution is a >30% salt solution. In another embodiment, thesolution is a <10% salt solution. In one embodiment, the salt solutionis NaCl (sodium chloride). In another embodiment, the salt solution isCaCl2, calcium chloride. The salt extraction step may be carried out ata specified temperature. In one embodiment, the salt extraction may becarried out at room temperature. In another embodiment, the saltextraction may be carried out below room temperature. In anotherembodiment, the salt extraction may be carried out at 4 degrees C. Inanother embodiment, the salt extraction may be carried out below 4degrees C.

Treatment to remove cell and cell debris may be carried out. Thematerials used to remove the cell debris may consist of enzymes,chemicals, detergents, salt solutions or hyperosmotic solutions. Aphysical agent, such as ultrasonic agitation, ultrasound, mechanicalagitation or electronic stimulation may be used as a decellularizationagent. The agents used to remove the cells and cell debris may consistof synthetic or natural materials. The tissue may be exposed to thesedecellularization agents for a specific time period. In one embodiment,the time period is 1 hour. In another embodiment, the time period is 1to 5 hours. In another embodiment, the time period is 5 to 10 hours. Inanother embodiment, the time period is 10 to 24 hours. In anotherembodiment, the time period is greater than 24 hours. In anotherembodiment, the time period is less than 1 hour. The tissue may beexposed to these decellularization agents at a specific temperature. Inone embodiment, the decellularization process may be carried out at roomtemperature. In another embodiment, the decellularization process may becarried out below room temperature. In another embodiment, thedecellularization process may be carried out at 4 degrees C. In anotherembodiment, the decellularization process may be carried out below 4degrees C. The tissue may be washed after the decellularization process.This wash may be performed using water, saline or other diluents. In oneembodiment, the decellularization agent is an enzyme. In anotherembodiment, the decellularization agent is sodium dodecyl sulfate (SDS).In another embodiment, the decellularization agent is DNAse. In anotherembodiment, the decellularization agent is RNAse. In another embodiment,the decellularization agent is Triton X. In another embodiment, thedecellularization agent is hypertonic NaCl. In another embodiment, thedecellularization agent is elastase. In another embodiment, thedecellularization agent is trypsin. In another embodiment, thedecellularization agent is a matrix metalloproteinase. A second saltextraction may be performed after the decellularization process. A rinsestep may be performed after the decellularization process or after thesalt extraction.

A step involving use of a citrate buffer may be used. The citratesolution may be used to extract additional collagen. The citrate buffermay be used at a pH=4. The citrate buffer may be placed in contact withthe tissue for as long as 48 hours. The citrate buffer step may becarried out at room temperature. The citrate buffer step may be carriedout at 4 degrees C. The tissue may be rinsed after addition of thecitrate to remove all or some of the citrate.

Ultracentrifugation may be used to process the tissue. Spin speeds ofover 1000 rpm may be used to pellet the tissue. This step may bealternated and repeated with wash steps with sterile solutions of acid,base, or neutral solutions.

An additional enzyme step may be used to break the collagen and/orglycosaminoglycans down into smaller fragments. An embodiment of thisenzyme step would use collagenase type I. An embodiment of this enzymestep would use collagenase type II. An embodiment of this enzyme stepwould use collagenase type III. An embodiment of this enzyme step woulduse a matrix metalloproteinase. An embodiment of this enzyme step woulduse matrix metalloproteinase-1. An embodiment of this enzyme step woulduse matrix metalloproteinase-13. An embodiment of this enzyme step woulduse pepsin. An embodiment of this enzyme step would use elastase. Anembodiment of this enzyme step would use trypsin. An embodiment of thisenzyme step would use an aggrecanase. An embodiment of this enzyme stepwould use chondroitinase.

A collagen extract as used herein refers to a high grade collagen slurrythat is useful in preparing collagen based scaffolds or tissuescaffolds. The term collagen extract is used interchangeably herein withthe terms slurry or collagen slurry. A collagen scaffold as used hereinis a solid or semi-solid/liquid material useful for implantation into ahuman subject or animal subject to repair damaged tissue and/or todeliver compounds and/or cells to the subject. The term collagenscaffold is used interchangeably herein with the terms sponge orcollagen sponge.

Detailed methods for preparing the collagen scaffold are provided below.The collagen slurry is prepared using the steps above. The collagencontent of the slurry is checked using the SIRCOL assay or similar assayfor collagen content. The slurry is then lyophilized to remove all waterand then resuspended with a measured amount of water, saline or otherdiluent to result in a slurry with the desired concentration ofcollagen. A strong acid or base can be added to the slurry to bring thepH to a desired level to inactivate any enzymes or chemicals used in theprocessing of the slurry that are desired to be inactivated beforeimplantation. Additional acid or base, or a buffer with a pK between 7and 8, can be then used to bring the pH of the solution to the desiredrange for implantation or combination with cells or proteins. Theosmolarity of the slurry can be adjusted to the desired range using asalt solution, or an acid or base. Once the slurry has the appropriatepH and osmolarity, it can be subjected to heat or cold to causeself-assembly or gelation of the collagen. After gelation,lyophilization of the scaffold can be used to produce a scaffold, spongeor powder. Alternatively, the solution can be maintained as a gel. In anembodiment, conditions are maintained to prevent collagen self-assemblyuntil after implantation of the collagen material into the joint.

Thus in some aspects, the invention involves a method for making acollagen scaffold. At its most basic the method involves steps ofpreparing a neutralized collagen slurry, heating the neutralizedcollagen slurry and freeze drying the heated neutralized collagenslurry. Preparing a neutralized collagen slurry can be achieved bymixing a collagen slurry with a neutralizing buffer. It may also involveadding a calcium containing solution. The heating step may be performed,for instance in a mold in a dry oven or in an incubator.

The methods of preparing a collagen extract involve preparing groundtissue by grinding freeze dried collagenous tissue that has beenisolated from a mammal. This can be performed by a homogenizer orsimilar device. A similar device is one that is useful for breaking thetissue into small pieces that can be effectively extracted. The methodsalso involve performing a first salt extraction on the ground tissue toproduce a salt extracted collagen, treating the salt extracted collagenwith a detergent solution, followed by an enzyme (in some embodimentselastase, in some embodiments RNase and/or Dnase, in some embodimentstrypsin, papain or one or more collagenase solutions) digestion and EDTAincubation to produce a collagen mixture, performing a second saltextraction on the collagen mixture and centrifuging the salt extractedmixture to produce a pellet, incubating the pellet with a citratebuffer, followed by acid solubilization and pepsin digestion.

The collagen scaffolds described herein may be used alone or incombination with three-dimensional (3-D) scaffolds or other traditionalrepair devices. The material provides a connection between the rupturedends of the ligament and fibers, or provides a replacement for a tornligament, after injury, and encourages the migration of appropriatehealing cells to form scar and new tissue and thus facilitating healingand regeneration.

It is intended that the use of the compositions and methods of thepresent invention involve the repair, replacement, reconstruction oraugmentation of specific tissue types. Articular injuries include bothintra-articular and extra-articular injuries. Intra-articular injuriesinvolve, for instance, injuries to meniscus, ligament and cartilage.Extra-articular injuries include, but are not limited to injuries to theligament, tendon or muscle. Thus, the methods of the invention may beused to treat injuries to the Anterior cruciate ligament (ACL), Lateralcollateral ligament (LCL), Posterior cruciate ligament (PCL), Medialcollateral ligament (MCL), Volar radiocarpal ligament, Dorsalradiocarpal ligament, Ulnar collateral ligament, Radial collateralligament, meniscus, labrum, for example glenoid labrum and acetabularlabrum, cartilage, for example, and other tissues exposed to synovialfluid after injury.

The injury being treated may be, for instance, a torn or rupturedligament. A ligament is a short band of tough fibrous connective tissuecomposed of collagen fibers. Ligaments connect bones to other bones toform a joint. A torn ligament is one where the ligament remainsconnected but has been damaged causing a tear in the ligament. The tearmay be of any length or shape. A ruptured ligament is one where theligament has been completely severed providing two separate ends of theligament. A ruptured ligament may provide two ligament ends of similaror different lengths. The rupture may be such that a ligament stump isformed at one end.

An example of a ruptured anterior cruciate ligament is described forexemplary purposes only. The anterior cruciate ligament (ACL) is one offour strong ligaments that connects the bones of the knee joint. Thefunction of the ACL is to provide stability to the knee and minimizestress across the knee joint. It restrains excessive forward movement ofthe lower leg bone, the tibia, in relation to the thigh bone, the femur,and limits the rotational movements of the knee. An anterior cruciateligament is ruptured such that it no longer forms a connection betweenthe femur bone and the tibia bone. The resulting ends of the rupturedACL may be of any length. The ends may be of a similar length, or oneend may be longer in length than the other.

The damaged or injured tissue is treated with the collagen scaffoldsdescribed herein which is typically a sterile solution of solubilizedcollagen. Solubilized collagen, as used herein, is enzyme solubilizedcollagen including one or more of Type I, II, III, IV, V, X collagen.Preferably the enzyme solubilized collagen is tropocollagen orAtelocollagen rather than fibrillar collagen in order to reduce theantigenicity of the material. The collagen is isolated from a tissuesource and mechanically minced and extracted as described above.Preferably the collagen is kept cold (4 deg C. or on ice) during storageand throughout parts of the preparation.

In one embodiment the solubilized collagen is Type I collagen. As usedherein the term, “Type I collagen” is characterized by two α1(I) chains,and one α 2(I) chains (heterotrimeric collagen). The α1 (I) chains areapproximately 300 nm long. Type I collagen is predominantly found inbone, skin (in sheet-like structures), and tendon (in rope-likestructures). Type I collagen is further typified by its reaction withthe protein core of another connective tissue component known as aproteoglycan. Type I collagen contains signaling regions that facilitatecell migration.

Natural sources of collagen may be obtained from animal or humansources. For instance, it may be derived from rat, pig, cow, or humantissue or tissue from any other species. Tendons, ligaments, muscle,fascia, skin, cartilage, tail, or any source of collagenous tissue areuseful. The material is then implanted into a subject of the same ordifferent species. The terms “xenogeneic” and “xenograft” refer to cellsor tissue which originates with or is derived from a species other thanthat of the recipient. Alternatively the collagen may be obtained fromautologous cells. For instance, the collagen may be derived from apatient's fibroblasts which have been cultured. The collagen may then beused in that patient or other patients. The terms “autologous” and“autograft” refer to tissue or cells which originate with or are derivedfrom the recipient, whereas the terms “allogeneic” and “allograft” referto cells and tissue which originate with or are derived from a donor ofthe same species as the recipient. The collagen may be isolated any timebefore surgery.

The collagen scaffold may be in a concentration of 1-100 mg/ml in thesolution. Such high concentrations of collagen are useful for producingviscosity levels that are desirable for the methods of the invention.Most commercially available collagen solutions are of lowerconcentrations. Higher concentrations can be made, for instance, usingthe methods described herein. In other embodiments the solubilizedcollagen solution has a concentration of 1 mg/ml to less than 5 mg/ml.

The collagen scaffold is sterile for in vivo use. The solution may besterilized and/or components of the solution may be isolated understerile conditions using sterile techniques to produce a sterilecomposition. The final desired properties of the composition may bedeterminative of how the solution is sterilized because somesterilization techniques may affect properties such as viscosity. Ifcertain components of the solution are not to be sterilized, i.e., thecollagen isolated from natural sources, the remaining components can becombined and sterilized before addition of the collagen, or eachcomponent can be sterilized separately. The solution can then be made bymixing each of the sterilized components with the collagen that has beenisolated using sterile techniques under sterile conditions.Sterilization may be accomplished, for instance, by autoclaving attemperatures on the order of about 115° C. to 130° C., preferably about120° C. to 125° C. for about 30 minutes to 1 hour. Gamma radiation isanother method for sterilizing components. Filtration is also possible,as is sterilization with ethylene oxide. Some embodiments includesterilization under low temperature conditions.

The collagen materials described herein may contain additionalcomponents, such as insoluble collagen, other extracellular matrixproteins (ECM), such as proteoglycans and glycosaminoglycans,fibronectin, laminin, entectin, decorin, lysyl oxidase, crosslinkingprecursors (reducible and non-reducible), elastin, elastin crosslinkprecursors, cell components such as, cell membrane proteins,mitochondrial proteins, nuclear proteins, cytosomal proteins, and cellsurface receptors, growth Factors, such as, PDGF, TGF, EGF, and VEGF,and hydroxyproline.

The methods described above include the addition of one or more buffersor solutions to produce the collagen extract and scaffold. The followingbuffers and solutions are useful in the methods of the invention:

Tris Buffer pH=7.5:

-   Tris_(MW)=121.14 and for 0.05M we'll need 6.057 g per liter of ultra    pure water (Milli-Q)    -   Add approx. 6.1 g of Tris in 800 ml of ultra pure water;    -   Stir at RT until dissolved;    -   pH to approx. 7.5 using HCl. Around 4 ml of 37% (˜12N) HCL; Add        3 ml and then add drop by drop with pH meter in until 7.5 is        reached    -   Complete to 1 liter with ultra pure water;

NaCl Solution:

-   20% is equivalent to 1.71M of NaCl solution (NaCl_(MW)=58.44)    -   Add 20 g of NaCl per 100 ml of Tris buffer;    -   Stir at RT until dissolved;    -   Filter.

Thrombin Solution:

-   1. Company: MP Biomwdicals    -   Tel: 800-883-9323    -   Cat #: 08820361    -   Lot #: 4869E    -   Quantity: 10× 1k unit    -   1,000 unit (1 vial) dilute in 40 mM CaCl2, depends on the        concentration of thrombin.        Preparation of L-Ascorbic Acid Phosphate Magnesium Salt        n-Hydrate Solution:-   Company: Wako-   Cat #: 013-12061-   MW=289.54-   Stock solution: 0.5 g in 50 ml of plain DMEM, dissolve, filter,    aliquot 1.5 ml.-   1.5 ml aliquot in 500 ml DMEM,-   Final concentration: 103.6 μM.-   Literature: 100 μM to 250 μM.

Preparation of MMP-3 Solution (Collagenase):

Concentration of 40.1 μg/ml needed, sold in concentration of 260 mg/ml(Chemicon #CC0315)

Use 0.001 ml of 260 mg/ml solution and add 249.999 ml of PBS

Preparation of MMP-1 Solution (Collagenase):

Concentration of 1.12 μg/ml, sold in concentration of 0.52 mg/ml(Chemicon #CC1031)

Use 19.27 ml of 0.52 mg/ml solution and add 230.73 ml of PBS.

Preparation of RNAse/DNAse Solution:

-   Add 476 mg MgCl₂ to 50 ml PBS and stir until dissolved    -   Add DNAse and RNAse into 5 ml tube and dissolve in MgCl PBS        solution, pour into MgCl PBS bottle, rinse out multiple times,        stir until dissolved

Preparation of MMP-2/MMP-9 Solution (Collagenase):

Concentration of 17.0 μg/ml, sold in concentration of 0.10 mg/ml(Chemicon #CC071).

Use 42.50 ml of 0.10 mg/ml solution and add 207.5 ml of PBS to make 250ml solution.

Preparation of Elastase Solution:

Concentration of 3.50 μg/ml, sold in 5 mg unit (Worthington #55K8226).

Dissolve 0.875 mg of Elastase in 250 ml of PBS.

Preparation of Papain Digest Solution:

-   Papain Digest buffer (100 mM sodium phosphate buffer/10 mM    Na₂EDTA/10 mM _(L)-cysteine/0.125 mg/mL papain) Prepare under    laminar flow hood. Cysteine and papain enzyme are unstable; use    fresh.    -   a. Prepare 40 mL PBE-10 mM cysteine; combine 40 mL PBE buffer        with 63.0 mg L-cysteine hydrochloride. Filter sterilize with a        0.22-μm syringe filter.    -   b. Prepare 20 mL papain digest buffer: transfer 20 mL sterile        PBE-cysteine to a fresh conical tube. Add papain enzyme using        sterile technique. Swipe rubber stopper of the papain solution        with ethanol, swirl to resuspend, and remove papain solution        with sterile 1-mL syringe and hypodermic needle to a sterile        eppendorf tube. Use a pipetteman to as 2.5 mg papain enzyme to        20 mL PBE-cysteine (add 100 μL if papain is 25 mg/mL).-   Add papain digest to gel, place tubes in heat block at 60° C. until    digested (>4 hours).

EDTA Solution

-   -   Add 20 mg EDTA to 100 ml PBS and stir until dissolved

-   Citrate buffer pH=4.0: Citric Acid—Sodium Citrate Buffer Solutions,    pH 3.0-6.21

-   Citric acid monohydrate, C₆H₈O₇•H₂O, M. wt. 210.14; 0.1M-solution    contains 21.01 g/l.

-   Trisodium citrate dihydrate, C₆H₈O₇Na₃•2H₂O, M. wt. 294.12;    0.1M-solution contains 29.41 g/l.

pH x ml 0.1M-citric acid y ml 0.1M-trisodium 3.0 82.0 18.0 3.2 77.5 22.53.4 73.0 27.0 3.6 68.5 31.5 3.8 63.5 36.5 4.0 59.0 41.0 4.2 54.0 46.04.4 49.5 50.5 4.6 44.5 55.5 4.8 40.0 60.0 5.0 35.0 65.0 5.2 30.5 69.55.4 25.5 74.5 5.6 21.0 79.0 5.8 16.0 84.0 6.0 11.5 88.5 6.2 8.0 92.0

Buffers: NaOH is 1M, HEPES is 0.1M

The above-described buffers and solutions are exemplary. The skilledartisan would recognize that some substitutions or adjustments could bemade to the buffers and solutions.

The buffers and solutions also may or may not include an antibiotic. Forinstance, the antibiotic may be penicillin/streptomycin. Alternativelyit may be a clinical antibiotic, which is used in human patients for thetreatment or prevention of diseases, such as any of those described inRemington's Pharmaceutical Sciences (Mack Publishing Co., Easton Pa.),which is hereby incorporated by reference.

The buffer may be a single component or it may be multiple componentsadded at the same time or different times. If the buffer is a singlecomponent it should have properties that enable it to produce a solutionhaving a desirable pH range and osmolarity. In some instances it isdesirable to have at least two buffer components, a collagen buffersolution and a neutralizing buffer. The collagen buffer solution may beused to prepare the collagen in a solution. In some instances theprepared collagen solution may be stored for extended periods of time.

In certain embodiments, the collagen solution is mixed with cells suchas platelets or white blood cells or red blood cells or stem cells orfibroblasts. In some embodiments, the cells are derived from the subjectto be treated. In other embodiments, the cells are derived from a donorthat is allogeneic to the subject.

In certain embodiments, platelets may be obtained as platelet richplasma (PRP). This component contains fibrin and platelets as well asother plasma proteins found in the blood. There may also be some whiteblood cells (WBC) and red blood cells (RBC) found in this preparation.Preferably the platelet concentration of PRP is at least 100K/ml, andpreferably over 300K/ml. For instance, the platelet concentration may beat least 1× what it is in the blood of the patient, and preferably 1.5×or greater In order to maintain the stability of the cells a physiologicpH (i.e., 6.2 to 7.6) and a physiologic plasma osmolarity (i.e., 280-360osms/kg) is used. In order to enhance the function of the PRP,preferably the PRP is used within 7 days of being drawn from the patientor donor. Often the PRP is isolated from the patient at time of surgery.Preferably it is stored at 20 to 37 deg C. (room temp to body temp).However, isolation and storage of the cells may be achieved by anymethods and for any length of time known in the art for maintaining theactivity of the active components.

In a non-limiting example, platelets may be isolated from a subject'sblood using techniques known to those of ordinary skill in the art. Asan example, a blood sample may be drawn into a tube containing ananticoagulant, and the subsequent solution centrifuged at 700 rpm for 20minutes and the platelet-rich plasma upper layer removed. Plateletdensity may be determined using a cell count as known to those ofordinary skill in the art. The platelet rich plasma may be mixed withcollagen and applied to the patient.

In a non-limiting example, white blood cells may also be isolated from asubject's blood using techniques known to those of ordinary skill in theart. As an example, a blood sample may be drawn into a tube containingan anticoagulant and centrifuged at 700 rpm for 20 minutes and the buffycoat containing white blood cells removed. WBC density may be determinedusing a cell count as known to those of ordinary skill in the art. TheWBCs can be mixed with collagen and applied to the patient. The collagensolution may also include any one or more of an anti-plasmin agent, anextracellular matrix (ECM) protein, other protein or enzyme inhibitors,antibodies to plasmin, antibodies to tissue plasminogen activator orurokinase plasminogen activator, non-toxic crosslinkers, calcium,dextrose or other sugars and cell nutrients in physiologicalconcentrations. Anti-plasmin agents include but are not limited toantifibrinolytic enzymes such as plasminogen inactivator, plasminogenbinding α₂ antiplasmin, non-plasminogen binding α₂ antiplasmin, α₂macroglobulin, α₂ plasmin inhibitor, α₂ antiplasmin, and thrombinactivatable fibrinolysis inhibitor. Other protein or enzyme inhibitorsinclude but are not limited to anti-enzymatic proteins includinginhibitors of collagenase, trypsin, matrix metalloproteinases, elastaseand hyaluronidase. The ECM is composed of fibrillar and non-fibrillarcomponents. The major fibrillar proteins are collagen and elastin. TheECM includes for instance, diverse combinations of collagens,fibrinogen, proteoglycans, elastin, hyaluronic acid, and variousglycoproteins including laminin, fibronectin, heparan sulfateproteoglycan, and entactin. Non-toxic crosslinkers include but are notlimited to tissue transglutaminases, lysyl oxidase, fibrin, fibronectin,and reducible and non-reducible crosslink precursor molecules.

The collagen solution, with or without any of the above-describedadditional components, may be stored as a liquid or gel material or maybe dried and stored as a powder. For instance, a collagen solution maybe lyophilized to produce a powder. The powder may then be reconstitutedin a buffer solution. Neutralizing agent may be present in thereconstitution buffer or may be added as a separate buffer or as salts.

The final collagen solution includes collagen, buffer and cells, such asPRP or WBCs. The components are mixed on a microscopic level, ratherthan layered. Preferably it has a pH of 7.4 and a minimum viscosity ofapproximately 1,000 centipoise. Preferably the viscosity is in the rangeof 1,000-200,000 centipoise.

While the degree of “solidness” may vary from application toapplication, generally speaking collagen solutions of the presentinvention will exhibit viscosities in the full range of from liquid togel-like to solid-like or even powder form. A collagen solution havingoptimal viscosity can be obtained directly from the source of collagen,depending on the concentration of the collagen. However, a collagensolution not having an optimal viscosity can be manipulated to createthe correct viscosity. The viscosity of a collagen solution may belowered by diluting the solution. The viscosity of a lower viscositycollagen solution may be increased to increase gelation. One method todo this is to lyophilize the collagen solution and rehydrate it in aspecific amount of water. Another method to do this is to stop thelyophilization process before it is completed, thus only removing partof the water from the gel and concentrating the collagen. Gelation isthe change in viscosity from a fluid-like composition to a solid orgel-like composition. Gelation or viscosity of a solution may also beincreased by adding one or more of the following: other ECM molecules,including but not limited to, insoluble collagen, fibrin, fibronectin,and cellulose; cell additions, including but not limited to, plateletsand fibroblasts; non-toxic crosslinking agents, including but notlimited to, tissue transglutaminases, lysyl oxidase, fibrin, andfibronectin; and other high viscosity materials with low osmolarity,including but not limited to, alginate and synthetic filler materials.

The term “repair material” as used herein refers to the finalformulation of collagen solution with cells to be delivered to thesubject.

The collagen solution or repair material may include additionalmaterials, such as growth factors, antibiotics, insoluble or solublecollagen, a cross-linking agent, thrombin, stem cells, a geneticallyaltered fibroblast, platelets, water, plasma, extracellular proteins anda cell media supplement. Alternatively the collagen solution or repairmaterial may exclude any of these components, and in particularthrombin. The additional materials may be added to affect cellproliferation, extracellular matrix production, consistency, inhibitionof disease or infection, tonicity, cell nutrients until nutritionalpathways are formed, and pH of the collagen solution or repair material.All or a portion of these additional materials may be mixed with thecollagen solution or repair material before or during implantation, oralternatively, the additional materials may be implanted proximate tothe defect area after the repair material is in place.

The repair material of the invention may be applied directly to thetissue alone or it may be used in combination with a tissue healingdevice such as a scaffold. A device or scaffold may be any shape that isuseful for implantation into a subject. The scaffold, for instance, canbe tubular, semi-tubular, cylindrical, including either a solid cylinderor a cylinder having hollow cavities, a tube, a flat sheet rolled into atube so as to define a hollow cavity, liquid, an amorphous shape whichconforms to that of the repair space, a “Chinese finger trap” design, atrough shape, or square. Other shapes suitable for the scaffold of thedevice as known to those of ordinary skill in the art are alsocontemplated in the invention.

The scaffold may be pretreated with the repair material prior toimplantation into a subject. For instance, the scaffold may be soaked ina repair material prior to or during implantation into a repair site.The repair material may be injected directly into the scaffold prior toor during implantation. The repair material may be injected within atubular scaffold at the time of repair.

In aspects of the invention, a device for use with the repair materialof the invention for repairing a damaged tissue includes a scaffoldand/or an anchor. A scaffold is capable of insertion into a repair siteand either forming a connection between the ends of a ruptured tissue,or forming around a torn tissue such that, in either case, the integrityand structure of the tissue is maintained. A scaffold is preferably madeof a compressible, resilient material which has some resistance todegradation by synovial fluid. Synovial fluid as part of normal jointactivity, naturally prevents clot formation. This fibrinolytic processwould result in the premature degradation of the scaffold and disruptthe healing process of the tissue. The material may be natural orsynthetic and may be either permanent or biodegradable material, such aspolymers and copolymers. The scaffold can be composed, for example, ofcollagen fibers, collagen gel, foamed rubber, natural material,synthetic materials such as rubber, silicone and plastic, ground andcompacted material, perforated material, or a compressible solidmaterial.

A scaffold that is capable of compression and expansion is particularlydesirable. For example, a sponge scaffold may be compressed prior to orduring implantation into a repair site. A compressed sponge scaffoldallows for the sponge scaffold to expand within the repair site.Examples of scaffolds useful according to the invention are found inU.S. Pat. No. 6,964,685 and US Patent Application Nos. 2004/0059416 and2005/0261736, the entire contents of each are herein incorporated byreference.

A scaffold may be a solid material such that its shape is maintained, ora semi-solid material capable of altering its shape and or size. Ascaffold may be made of expandable material allowing it to contract orexpand as required. The material can be capable of absorbing plasma,blood, other body fluids, liquid, hydrogel, or other material thescaffold either comes into contact with or is added to the scaffold.

A scaffold material may incorporate therapeutic proteins including, butnot limited to, hormones, cytokines, growth factors, clotting factors,anti-protease proteins (e.g., alpha1-antitrypsin), angiogenic proteins(e.g., vascular endothelial growth factor, fibroblast growth factors),antiangiogenic proteins (e.g., endostatin, angiostatin), and otherproteins that are present in the blood, bone morphogenic proteins(BMPs), osteoinductive factor (IFO), fibronectin (FN), endothelial cellgrowth factor (ECGF), cementum attachment extracts (CAE), ketanserin,human growth hormone (HGH), animal growth hormones, epidermal growthfactor (EGF), interleukin-1 (IL-1), human alpha thrombin, transforminggrowth factor (TGF-beta), insulin-like growth factor (IGF-1), plateletderived growth factors (PDGF), fibroblast growth factors (FGF, bFGF,etc.), and periodontal ligament chemotactic factor (PDLGF), fortherapeutic purposes. A lyophilized material is one that is capable ofswelling when liquid, gel or other fluid is added or comes into contactwith it.

A device for implantation may also include one or more anchors. Ananchor is a device capable of insertion into a bone or tissue such thatit forms a stable attachment to the bone or tissue. In some instancesthe anchor is capable of being removed from the bone if desired. Ananchor may be conical shaped having a sharpened tip at one end and abody having a longitudinal axis. The body of an anchor may increase indiameter along its longitudinal axis. The body of an anchor may includegrooves suitable for screwing the anchor into position. An anchor mayinclude an eyelet at the base of the anchor body through which one ormore sutures may be passed. The eyelet may be oval or round and may beof any size suitable to allow one or more sutures to pass through and beheld within the eyelet.

An anchor may be attached to a bone or tissue by physical or mechanicalmethods as known to those of ordinary skill in the art. An anchorincludes, but is not limited to, a screw, a barb, a helical anchor, astaple, a clip, a snap, a rivet, or a crimp-type anchor. The body of ananchor may be varied in length. Examples of anchors, include but are notlimited to, IN-FAST™ Bone Screw System (Influence, Inc., San Francisco,Calif.), IN-TAC™ Bone Anchor System (Influence, Inc., San Francisco,Calif.), Model 3000 AXYALOOP™ Titanium Bone Anchor (Axya Medical Inc.,Beverly, Mass.), OPUS MAGNUM® Anchor with Inserter (Opus Medical, Inc.,San Juan Capistrano, Calif.), ANCHRON™, HEXALON™, TRINION™ (allavailable from Inion Inc., Oklahoma City, Okla.) and TwinFix ABabsorbable suture anchor (Smith & Nephew, Inc., Andover, Mass.). Anchorsare available commercially from manufacturers such as Influence, Inc.,San Francisco, Calif., Axya Medical Inc., Beverly, Mass., Opus Medical,Inc., San Juan Capistrano, Calif., Inion Inc., Oklahoma City, Okla., andSmith & Nephew, Inc., Andover, Mass.

An anchor may be composed of a non-degradable material, such as metal,for example titanium 316 LVM stainless steel, CoCrMo alloy, or Nitinolalloy, or plastic. An anchor is preferably bioabsorbable such that thesubject is capable of breaking down the anchor and absorbing it.Examples of bioabsorbable material include, but are not limited to,MONOCRYL (poliglecaprone 25), PDS II (polydioxanone), surgical gutsuture (SGS), gut, coated VICRYL (polyglactin 910, polyglactin 910braided), human autograft tendon material, collagen fiber, POLYSORB,poly-L-lactic acid (PLLA), polylactic acid (PLA), polysulfone,polylactides (Pla), racemic form of polylactide (D,L-Pla),poly(L-lactide-co-D,L-lactide), 70/30 poly(L-lactide-co-D,L-lactide),polyglycolides (PGa), polyglycolic acid (PGA), polycaprolactone (PCL),polydioxanone (PDS), polyhydroxyacids, and resorbable plate material(see e.g. Orthopedics, October 2002, Vol. 25, No. 10/Supp.). The anchormay be bioabsorbed over a period of time which includes, but is notlimited to, days, weeks, months or years.

The scaffold may also be secured in place using sutures passed throughbone tunnels or both and anchored on the outer surfaces of the boneusing a button or small plate. Examples of these buttons are commonshirt buttons or buttons made specifically for medical applications, forexample the ENDOBUTTON (Smith and Nephew, Andover Mass.). In oneembodiment, sutures are passed through bone tunnels in both femur andtibia, creating a suture stent to secure the scaffold in the knee.

A suture is preferably bioabsorbable, such that the subject is capableof breaking down the suture and absorbing it, and synthetic such thatthe suture may not be from a natural source. Examples of suturesinclude, but are not limited to, VICRYL™ polyglactin 910, PANACRYL™absorbable suture, PDS® polydioxanone suture and PROLENE® polypropylenesuture. Sutures are available commercially from manufacturers such asMITEK PRODUCTS division of ETHICON, INC. of Westwood, Mass.Alternatively, the suture is non-absorbable and is considered apermanent implant. In another embodiment, the suture is non-absorbableand is able to be released at a specific time point. Examples ofnon-absorbable sutures include ETHIBOND® EXCEL polyester suture,stainless steel wire or Teflon or nylon materials.

A staple is a type of anchor having two arms that are capable ofinsertion into a bone or tissue. In some instances, the arms of thestaple fold in on themselves when attached to a bone or in someinstances when attached to other tissue. A staple may be composed ofmetal, for example titanium or stainless steel, plastic, or anybiodegradable material. A staple includes but is not limited to linearstaples, circular staples, curved staples or straight staples. Staplesare available commercially from manufacturers such as Johnson & JohnsonHealth Care Systems, Inc. Piscataway, N.J., and Ethicon, Inc.,Somerville, N.J. A staple may be attached using any staple device knownto those of ordinary skill in the art, for example, a hammer and staplesetter (staple holder).

The device may be inserted into a repair site of the ruptured or torntissue. A repair site is the area around a ruptured or torn tissue intowhich the material of the invention may be inserted. A device may beplaced into a repair site area during surgery using techniques known tothose of ordinary skill in the art. If a scaffold is used in themethods, the scaffold can either fill the repair site or partially fillthe repair site. A scaffold can partially fill the repair site wheninserted and expand to fill the repair site in the presence of blood,plasma anticoagulated blood, anticoagulated blood products or otherfluids either present within the repair site or added into the repairsite, such as the repair material.

The scaffold may be positioned in combination with a surgical technique.For instance, a hole may be drilled into a bone at or near a repair siteof a ruptured or torn tissue and the scaffold attached by a suturethrough the hole to the bone. A bone at or near a repair site is onethat is within close proximity to the repair site and can be utilizedusing the methods and devices of the invention. For example, a bone ator near a repair site of a torn anterior cruciate ligament is a femurbone and/or a tibia bone. A hole can be drilled into a bone using adevice such as a Kirschner wire (for example a small Kirschner wire) anddrill, or microfracture pics or awls.

A hole may be drilled into a bone on the opposite side to the repairsite. A suture may be passed through the hole in the bone and attachedto the bone. A scaffold is attached to the suture to secure the scaffoldbetween the bone and an end of a ruptured tissue. A ruptured tissueprovides two ends of the tissue that were previously connected. Ascaffold may be attached to one or both ends of a ruptured tissue by oneor more sutures. A suture may be attached to a second bone site at ornear the repair site. The suture may be attached to the second boneusing a second anchor.

In a typical arthroscopic procedure, for instance of the ACL, thesurgeon prepares the patient for surgery by insufflating the patient'sknee with sterile saline solution. Several cannulas are inserted intothe knee and used as entry portals into the interior of the knee. Aconventional arthroscope is inserted through one of the cannulas so thatthe knee may be viewed by the surgeon remotely.

In surgical reconstruction of a tissue such as ACL the surgeon may drilla tibial tunnel and a femoral tunnel in accordance with conventionalsurgical techniques using conventional surgical drills and drill guides.A replacement anterior cruciate ligament graft is then prepared andmounted in the tibial and femoral tunnels, and secured usingconventional techniques and known devices in order to complete the kneereconstruction.

The repair material may also be used in combination with a graft, suchas an ACL graft. Several types of ACL grafts are available for use bythe surgeon in ACL reconstruction. The grafts may be autografts that areharvested from the patient, for example patellar bone-tendon-bonegrafts, or hamstring grafts. Alternatively, the grafts can bexenografts, allografts, or synthetic polymer grafts. Allografts includeligamentous tissue harvested from cadavers and appropriately treated anddisinfected, and preferably sterilized. Xenografts include harvestedconnective tissue from animal sources such as, for example, porcinetissue. Typically, the xenografts must be appropriately treated toeliminate or minimize an immune response. Synthetic grafts includegrafts made from synthetic polymers such as polyurethane, polyethylene,polyester and other conventional biocompatible bioabsorbable ornonabsorbable polymers and composites, such as the scaffolds describedherein.

The repair material is applied to a subject. The application to thesubject involves surgical procedures. The following is an example of asurgical procedure which may be performed using the methods of theinvention. The affected extremity is prepared and draped in the standardsterile fashion. A tourniquet may be used if indicated. Theintra-articular lesion is identified and defined, the tissue ends arepretreated, either mechanically or chemically, and if a scaffold isbeing used, the scaffold is introduced into the tissue defect. If thescaffold has not been pre-soaked in any additional components designedto facilitate repair, for example platelets, growth factors or othercells, or if more repair material is desired, then the repair materialis added to the scaffold. The scaffold may be reinforced by placement ofsutures or clips. If no scaffold is used the tissue defect is coateddirectly with repair material. The post-operative rehabilitation isdependent on the joint affected, the type and size of lesion treated,and the tissue involved.

The methods of the invention may be achieved using arthroscopicprocedures. Standard arthroscopy equipment may be used. Initially,diagnostic arthroscopy may be performed to identify the appropriaterepair site. If a scaffold is used it should be compressible to allowintroduction through arthroscopic portals, incisions and equipment. Therepair material can be placed in the repair site by direct injection.After the procedure the arthroscopic portals can be closed and a steriledressing placed.

A subject includes, but is not limited to, any mammal, such as human,non-human primate, mouse, rat, dog, cat, horse or cow. In certainembodiments, a subject is a human.

The materials used in the invention are preferably biocompatible,pharmaceutically acceptable and sterile. As used herein, the term“biocompatible” refers to compositions (e.g. cells, tissues, matrices,etc.) that do not substantially disrupt the normal biological functionsof other compositions to which they contact. In selected embodiments,the present invention also contemplates biocompatible materials that areboth biodegradable and non-biodegradable.

As described above, each of the components of the repair material may beprepared sterilely. If however, one or more components is not retrievedor processed in a sterile manner then it can be sterilized prior toapplication to the subject. For instance the material (preferablywithout the cells) may be sterilized after production using gammairradiation, ethanol, autoclave sterilization or other knownsterilization methods.

As used herein, the term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the scaffold material or repair material. Theterm “physiologically acceptable” refers to a non-toxic material that iscompatible with a biological system such as a cell, cell culture,tissue, or organism. The characteristics of the carrier will depend onthe route of administration. Physiologically and pharmaceuticallyacceptable carriers include diluents, fillers, salts, buffers,stabilizers, solubilizers, and other materials which are well known inthe art. The term “carrier” denotes an organic or inorganic ingredient,natural or synthetic, with which the scaffold material is combined tofacilitate the application. The components of the pharmaceuticalcompositions also are capable of being co-mingled with the device of thepresent invention, and with each other, in a manner such that there isno interaction which would substantially impair the desiredpharmaceutical efficacy.

In some embodiments the repair material composition is injectable.Injectable compositions may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Pharmaceuticalformulations for injection may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the materials may also containsuitable stabilizers.

The collagen solution may be in the form of a liquid, gel or solid,prior to addition of the cells. Once the cells are added, the repairmaterial will begin to increase in gelation for application to the body.If the collagen solution is a liquid or gel the cells may be directlyadded to the solution.

Alternatively, the collagen solution may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use. Neutralization agent may be added before or afterreconsistution. After the powder is reconsituted it is mixed with cellsto form the repair material.

As used herein, the term “gel” refers to the state of matter betweenliquid and solid. As such, a “gel” has some of the properties of aliquid (i.e., the shape is resilient and deformable) and some of theproperties of a solid (i.e., the shape is discrete enough to maintainthree dimensions on a two dimensional surface.). A gel may be providedin pharmaceutical acceptable carriers known to those skilled in the art,such as saline or phosphate buffered saline. Such carriers may routinelycontain pharmaceutically acceptable concentrations of salt, bufferingagents, preservatives, compatible carriers and optionally othertherapeutic agents.

An example of a gel is a hydrogel. A hydrogel is a substance that isformed when an organic polymer (natural or synthetic) is crosslinked viacovalent, ionic, or hydrogen bonds to create a three-dimensionalopen-lattice structure which entraps water molecules to form a gel. Apolymer may be crosslinked to form a hydrogel either before or afterimplantation into a subject. For instance, a hydrogel may be formed insitu, for example, at the repair site. In certain embodiments, therepair material forms a hydrogel within the repair site upon exposure tobody temperatures.

The repair material, including the collagen solution and the cells willbegin to set once it is created. The setting process can be delayed bymaintaining cold temperatures or it may be accelerated by warming themixture. In certain embodiments, a quick set composition of the repairmaterial is provided. The quick set composition is capable of forming aset scaffold within 10 minutes of mixture when the material is exposedto temperatures of greater than 30° C. In some embodiments formation ofthe scaffold takes approximately 5 minutes at such temperatures. Thequick set composition is achieved by preparing the collagen solution atconcentrations and viscosities as described herein. The quick set naturecan be further enhanced by the addition of non-toxic cross linkingagents. Such compositions should be applied quickly to the tissue defectto sufficiently set before closure of the defect and surgery area.

The invention also includes in some aspects kits for making the collagenmaterial. A kit may include one or more containers housing thecomponents of the invention. The kit may be designed to facilitate useof the methods described herein and can take many forms. Each of thecompositions of the kit, where applicable, may be provided in liquidform (e.g., in solution), or in solid form, (e.g., a dry powder). Incertain cases, some of the compositions may be constitutable orotherwise processable (e.g., to an active form), for example, by theaddition of a suitable solvent or other species (for example, water or acell culture medium), which may or may not be provided with the kit. Asused herein, “instructions” can define a component of instruction and/orpromotion, and typically involve written instructions on or associatedwith packaging of the invention. Instructions also can include any oralor electronic instructions provided in any manner such that a user willclearly recognize that the instructions are to be associated with thekit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet,and/or web-based communications, etc. The written instructions may be ina form prescribed by a governmental agency regulating the manufacture,use or sale of pharmaceuticals or biological products, whichinstructions can also reflects approval by the agency of manufacture,use or sale for human administration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit. Thekit may include a container housing the collagen source. The collagenmay be in the form of a liquid, gel or solid (powder). The collagen maybe prepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have buffer solution premixed prepared sterilelyor in the form of salts.

The kit may be sterilized after the accessories are added, therebyallowing the individual accessories in the container to be otherwiseunwrapped. The kits can be sterilized using any appropriatesterilization techniques, such as radiation sterilization, heatsterilization, or other sterilization methods known in the art.

The kit may include disposable components supplied sterile in disposablepackaging. The kit may also include other components, depending on thespecific application, for example, containers, cell media, salts,buffers, reagents, syringes, needles, etc.

In another example, a collagen solution can be lyophilized first andthen rehydrated. The collagen slurry thus produced can be neutralized bya method known in the art (e.g., using a HEPES buffer) and thenincubated under suitable conditions to allow for gelation. The collagengel thus formed can be lyophilized to produce a collagen sponge.Afterwards, the collagen sponge can be soaked in a calcium solutionhaving a suitable calcium concentration (e.g., those described herein)for a suitable period and then lyophilized to produce acalcium-containing collagen material. In another embodiment, thestructural member and the biomaterials described herein aresubstantially free of thrombin. In another embodiment, no non-autologousthrombin is added to the biomaterial before, during, or afterimplantation. In another embodiment, thrombin from any source is addedto the biomaterial before, during, or after implantation. In anotherembodiment, the only thrombin that is added to the biomaterial before,during, or after implantation is that found in the autologous blood orplasma comprising the implanted or injected material.

In some embodiments, the biomaterial described herein (e.g., collagenmaterials or ECM scaffolds) are substantially free of one or more of thefollowing cell components: nucleic acid (DNA and/or RNA),glycosaminoglycan (GAG), phospholipid, active pepsin, and active virus.Such compositions can be prepared by treating the biomaterial, thestructural member contained therein, or the solution for preparing thebiomaterial/structural member to remove DNA, DNA fragments, RNA and RNAfragments, cells, fragments of cell membrane, cell components, and/or tominimizes the incorporation of endotoxins. The biomaterials, structuralmembers, and/or solutions for preparing such can also be treated toinactivate pepsin and remove/inactivate viruses. Such treatments can beperformed following methods known in the art and/or those descriedherein (see Examples 7 and 8 below).

In some embodiments, the composite of structural members andbiomaterials described herein (e.g., collagen materials or ECM scaffoldsand/or a bio-active agent added to the collagen or ECM materials) aresubstantially free of one or more of the following components: thrombin,non-autologous cellular components, active pepsin, and active virus.Such compositions can be prepared by treating the biomaterial, thestructural member contained therein, or the solution for preparing thebiomaterial/structural member to remove thrombin, DNA, DNA fragments,RNA and RNA fragments, cells, fragments of cell membrane, cellcomponents, and/or to minimizes the incorporation of endotoxins orviruses. The biomaterials, structural members, and/or solutions forpreparing such can also be treated to inactivate pepsin andremove/inactivate viruses. Such treatments can be performed followingmethods known in the art and/or those described herein (see Examples 7and 8 below).

For example, methods for removing cell components can involve the use ofdetergents, including SDS, EDTA, TritonX, polyethylene glyco, citrate,and sodium deoxycholate. These methods may also include the use of asurfactant. In addition, these methods may involve the use of enzymes,including trypsin, collagenase, elastase, DNAse and RNAse, ribonuclease,deoxyribonuclease, alpha-galactosidase, and other enzymes which candegrade cell membranes, receptors, or other cellular components. Thesemethods may also include physical processes, including ultrasound,electron beam irradiation and gamma irradiation.

For each of these solutions containing one or more agents noted above,concentrations of solutions from 0.001% to 50% may be used. Preferredembodiments are for solutions in the range of 0.001 to 1.0%. Otherpreferred embodiments are for solutions in the range of 0.1 to 10.0%.Other solutions may be used in concentrations of IU/ml, for example,DNAse may be used in a concentration of 150 IU/ml.

In some examples, the pepsin in a biomaterial as described herein can beinactivated by bringing the pH of an ECM slurry such as a collagenslurry above 4.0 using a strong base such as NaOH or LiOH or KOH. Otherbases include Ba(OH)₂ and Sr(OH)₂ can also be used to increase the pH ofthe solution and inactivate the pepsin. To inactivate the pepsin, asuitable volume of a suitable concentration of the strong base is addeddropwise to the pepsin-containing slurry and the pH is recorded. Thisprocess is repeated until the pH of the slurry is above 4.0.Alternatively, the exact amount of the strong base that needs to beadded is calculated based on the hydrogen ion content in the volume ofslurry that needs to be counteracted by the strong base to raise the pHof the solution significantly and then that exact volume is measured andthe pH is checked to ensure it is above 4.0. In other embodiments, theprocess is done in either of these two ways, but additional strong baseis added until the pH reaches 6.0. In other embodiments, the process isdone in either of these two ways, but additional strong base is addeduntil the pH reaches 7.0 or greater. In other embodiments, the processis done in either of these two ways, but additional strong base is addeduntil the pH reaches 8.0 or greater. In other embodiments, the processis done in either of these two ways, but additional strong base is addeduntil the pH reaches 9.0 or greater. In other embodiments, the processis done in either of these two ways, but additional strong base is addeduntil the pH reaches 10.0 or greater. In other embodiments, the processis done in either of these two ways, but additional strong base is addeduntil the pH reaches 11.0 or greater. In other embodiments, the processis done in either of these two ways, but additional strong base is addeduntil the pH reaches 5.0 or greater.

Once the slurry reaches its target pH range, the solution is kept therefor a specific amount of time. This time may be between 10 seconds and 1week. In a preferred embodiment, the time is between 1 and 10 minutes.In another preferred embodiment, the time is between 10 and 30 minutes.In another preferred embodiment, the time is between 10 and 60 minutes.

After inactivation of the pepsin, the pH of the slurry is returned to apH between 7.0 and 8.0 by the addition of a buffer with a pK of between7 and 8, such as a buffer containing TAPSO, HEPES, TES, MOPS, CacodylateSSC or Succinic acid. Alternatively, phosphate buffered saline may beused, or K₂HPO₄. Any combination of a weak acid and its conjugate base,or a weak base and its conjugate acid may be used. A buffer of carbonicacid and bicarbonate may also be used. Blood or plasma containingcarbonic acid and bicarbonate may be used. A universal buffer, such asthat using citric acid and Na2HPO4 (McIlvaine's buffer solutions) mayalso be used in proportions that yield a buffer range of pH between 7and 8.

Upon treatment as described above, the biomaterial described herein issubstantial free of one or more of nucleic acid (DNA and/or RNA),glycosaminoglycan (GAG), phospholipid, active pepsin, and active virus.In some examples, the nucleic acid and/or phospholipid content in thebiomaterial (e.g., dry weight) described herein is less than 20% of thatin a native tissue such as dermis (e.g., less than 15%, 10%, 5%, or 1%).In other examples, the content of GAG in the biomaterial is less than50% (e.g., 40%, 30%, 20%, 15%, 10%, or 5%) of the total dry weight ofthe biomaterial. In other examples, the level of active pepsin in thebiomaterial described herein is less than 1000 ug/ml, e.g., less than500 ug/ml, 200 ug/ml, or 100 ug/ml.

In some examples, viruses can be inactivated by ethylene oxidesterilization, e-beam sterilization, or gamma irradiation.

Medical Uses of Biomaterials

The biomaterials described herein, including collagen materials such ascollagen gels and collagen sponges, and ECM scaffolds, can be used toprevent and/or minimize progression of injuries to the anterior cruciateligament, the meniscus, labrum, cartilage, and other tissues exposed tosynovial fluid after injury. They also can be used to alleviate and/orreduce the risk for developing arthritis (e.g., osteoarthritis), such aspost-traumatic arthritis.

In some embodiments, the biomaterials described herein (e.g., the ECMscaffolds such as collagen scaffolds) are designed for use in anarthroscopic surgery with arthroscopic equipment. The scaffold can becompressible to allow introduction through arthroscopic portals andequipment. When desired, the scaffold can also be pre-treated inantibiotic solution or sterilization via a routine method prior toimplantation. When a collagen-based scaffold is used in the treatmentdescribed herein, the affected extremity is prepared and draped in thestandard sterile fashion. A tourniquet may be used if indicated.Standard arthroscopy equipment may be used. After diagnostic arthroscopyis performed, and an intra-articular lesion identified and defined,tissues desired for protection are pretreated, either mechanically orchemically, and the scaffold introduced into the joint. The scaffold isthen bonded to the surrounding tissue by creating chemical or mechanicalbonds between the tissue proteins and the scaffold biologic agent. Thiscan be done by the addition of a chemical agent or a physical agent suchultraviolet light, a laser, or heat, the scaffold may be reinforced byplacement of sutures or clips. The arthroscopic portals can be closedand a sterile dressing placed. The post-operative rehabilitation isdependent on the joint affected, the type and size of lesion treated,and the tissue involved.

EXAMPLES Example 1 Terminal Sterilization Study

Methods: In this study, an extracellular matrix (ECM) scaffold preparedaccording to the methods of the invention was terminally sterilizedusing standard E-beam and EO sterilization. Briefly, the scaffolds wereprepared by lyophilizing bovine connective tissue, washing the tissue,digesting in pepsin and then lyophilizing in a mold to make acylindrical scaffold. The samples were placed inside glass vials. Theglass vials were purged by Nitrogen in order to reduce the amount of theoxygen in them. The glass vials were then sealed and clamped as shown inFIG. 1 and stored on dry ice throughout the sterilization procedure.

E-Beam: For the e-beam groups, the ECM scaffolds were placed in glassvials that were capped and sealed using plastic stoppers and crimpedusing an aluminum clamping system. The samples were then subjected to 15kGy E-Beam sterilization process by Sterigenics (San Diego, Calif.). An“e-beam” control group of scaffolds was shipped to Sterigenics but notsterilized and shipped back for testing.

-   The following conditions were used in the study for the 15 kGy    E-beam process:

1. Temperature; sterilization under frozen condition on dry ice

2. Presence of Oxygen; the vial containing ECM Scaffold will be free ofoxygen

(Nitrogen Purged)

3. Dosage; 15 kGy

The packaging of the samples was similar to the image shown in FIG. 1;glass vials with rubber stoppers which will be sealed on top viaaluminum sealing system.

EO: For the ethylene oxide groups, lyophilized ECM scaffolds were placedinto gas permeable pouches and subjected to EO sterilization. TheEthylene concentration used was 735 mg/l for at least 4.75 hours. Eachpouch was marked with the EO sterilization indicator to confirm the EOsterilization. The packaging of the samples was similar to image shownin FIG. 1.

-   Table 1 provides a summary of the study design.

TABLE 1 Study design for ECM scaffold sterilization using E-Beam and EOsterilization. E-Beam Dose Condition # of Samples ID [kGy] OutcomeMeasure Sterile process ECM 3 of 1, 2, 3 15 1. Culture in media in anincubator for Scaffold in sealed glass OD = 22 mm 7 days (n = 6; twofrom each scaffold). vial, purged by nitrogen, Length = 30 Cut 2 mmlength from the bottom of frozen, and subjected to mm each scaffoldprior to other testing) E-Beam sterilization 2. Passage a straight Keithneedle while kept on dry ice through the scaffold in four parallelplaces during sterilization without the scaffold fracturing (n = 3;prior to all tests except culture samples removed for #1 before thisstep) 3. Resistance to digestion by collagenase, elastase and plasminable to (n = 6; two new samples from each scaffold) 4. A 22 mm dia × 10mm length of ECM scaffold able to absorb 3 cc of blood (n = 3)Sterile-processed ECM 3 of 4, 5, 6 N/A 1. Culture in media in anincubator for Scaffold in sealed glass OD = 22 mm 7 days (n = 6; twofrom each scaffold). vial, purged by nitrogen, Length = 30 Cut 2 mmlength from the bottom of frozen shipped on dry ice mm each scaffoldprior to other testing) to the sterilization 2. Passage a straight Keithneedle facility and back, similar through the scaffold in four parallelto top row samples. places without the scaffold fracturing (n = 3; priorto all tests except culture samples removed for #1 before this step) 3.Resistance to digestion by collagenase, elastase and plasmin able to (n= 6; two new samples from each scaffold) 4. A 22 mm dia × 10 mm lengthof ECM scaffold able to absorb 3 cc of blood (n = 3) Sterile-processedECM 3 of 7, 8, 9 N/A 1. Culture in media in an incubator for Scaffoldsamples kept at OD = 22 mm 7 days (n = 6; two from each scaffold). CHBLength = 30 Cut 2 mm length from the bottom of mm each scaffold prior toother testing) 2. Passage a straight Keith needle through the scaffoldin four parallel places without the scaffold fracturing (n = 3; prior toall tests except culture samples removed for #1 before this step) 3.Resistance to digestion by collagenase, elastase and plasmin able to (n= 6; two new samples from each scaffold) 4. A 22 mm dia × 10 mm lengthof ECM scaffold able to absorb 3 cc of blood (n = 3) ECM Scaffold 3 of10, 11, Low- 1. Culture in media in an incubator for packaged as shownin OD = 22 mm 12 temp 7 days (n = 6; two from each scaffold). FIG. 1 andLength = 30 EO Cut 2 mm length from the bottom of subjected to EO mmeach scaffold prior to other testing) sterilization 2. Passage astraight Keith needle through the scaffold in four parallel placeswithout the scaffold fracturing (n = 3; prior to all tests exceptculture samples removed for #1 before this step) 3. Resistance todigestion by collagenase, elastase and plasmin able to (n = 6; two newsamples from each scaffold) 4. A 22 mm dia × 10 mm length of ECMscaffold able to absorb 3 cc of blood (n = 3)

-   Following the sterilization processes, the ECM Scaffold was    subjected to a series of tests alongside the control non-sterile ECM    Scaffold to determine whether the sterilization process caused any    changes to the ECM Scaffold characteristics. The test included:

1) The scaffolds were incubated in culture in media for 7 days. Noantibiotics were included in the media. The samples used for the culturetest were cut in a laminar hood using sterile technique before anymanipulation on the samples. A disc of 1 to 2 mm thickness was enoughfor each samples.

2) Passage of a straight Keith needle through the scaffold in fourparallel places by hand and assessing any scaffold fracturing.

3) Collagenase digestion (250 ug/ml in PBS) times of 24 to 48 hours,Elastase digestion (100 ug/ml in PBS) times of 14 to 21 days, andPlasmin Digestion (40 ug/ml in water) digestion times of 7 to 14 days.One ml of enzyme solution will be used for each sample in separate wellsof a 6 well plate. This is assuming the control non-sterilized ECMscaffold has times of 24 hours, 14 days and 7 days for collagenase,elastase and plasmin respectively. Sample size for each digestion assaywas 8 mm diam, 7 mm height created by making a 7 mm slice of the largerscaffolds and then using an 8 mm sterile punch to get individualsamples.

4) Absorption of blood; a 22 mm diameter×10 mm length of ECM scaffoldshould be able to absorb 3 cc of blood.

-   Results: The results obtained from the sterilized samples were    compared to those obtained from the non-sterilized ECM Scaffold and    control samples.

1. Culture Study:

4 out of six of the sterile processed control scaffolds showed a colorchange in the media after 14 days. The terminal sterilized specimens didnot show any change. None of the wells had any growth of bacteria orfungus after 14 days that was grossly visible. No organisms were visiblewith microscopic examination of the culture wells for any of thescaffolds. The results demonstrate the efficacy of the sterilizationtechniques.

2. Keith Needle Study:

The panels are shown from left to right as follows: Ebeam/EbeamControl/EO/Control. All four scaffolds showed no signs of breakage.

3. Enzyme Degradation Study:

In each instance, the scaffolds sterilized using Ebeam had slightlyfaster digestion rates. In general all the samples were very resistant.

4. Blood Absorption Study:

Absorption time was significantly prolonged in the EO group. All groupsabsorbed the total of 1 ml of whole blood. Loss of height duringabsorption was increased in the Ebeam group.

Gross Appearance: The Ebeam scaffolds showed a slight yellowdiscoloration. Whereas the control Ebeam scaffolds had no change. The EOscaffold had very slight yellow discoloration. From left to right:Ebeam/Ebeam Control/EO/Control.

Example 2 Evaluation of DNA Content/RNA Content/Cell Fragment Content inScaffolds

Scaffold were treated with various chemicals and enzymes to lower theDNA, GAG and Phospholipid content. As described in detail below it waspossible to significantly reduce the DNA, GAG and Phospholipid contentof the scaffolds.

In this experiment, tissues from 8 bovine knees were collected. Thetotal wet weight of the harvested tissues was 166.5 g. The tissues werelyophilized until dry and then homogenized. The homogenized tissue wasthen divided into 16 samples with dry weights. The dry tissue sampleswere then rinsed in 2% antibiotic solution overnight.

Start of Differentiation:

-   Following the antibiotic rinse, the samples were divided into 16    different treatment groups as noted below.    Summary of Sample Groups: Differences only are Noted in Table 2.

Sample 1: The tissue was rinsed with NaCl solution followed by ultrapurewater three times, and then washed with citrate buffer with a pH=4.0 for72 hours. The samples were ultracentrifuged and treated with pepsin and0.01NHCl. The amount of pepsin and HCl was determined using the wetweight of the sample. The remaining sample was tested.

Sample 2: The tissue was rinsed with NaCl solution followed by ultrapurewater three times. The samples were ultracentrifuged and treated withpepsin and HCl. The amount of pepsin and HCl was determined using thewet weight of the sample. The remaining sample was tested.

Sample 3: The tissue was rinsed with NaCl solution followed by ultrapurewater. The sample was then treated with sterile RNase A (100 μg/mL) andDNase I (150 IU/mL) with 10 mmolMgCl2, in 0.05M Tris-Buffer (pH=7.5) andthen washed with citrate buffer with a pH=4.0 for 72 hours. The tissuewas then again rinsed with NaCl solution. The samples wereultracentrifuged and treated with pepsin and HCl. The amount of pepsinand HCl was determined using the wet weight of the sample. The remainingsample was tested.

Sample 4: The tissue was rinsed with NaCl solution followed by ultrapurewater. The sample was then treated with sterile RNase A (100 μg/mL) andDNase I (150 IU/mL) with 10 mmolMgCl2, in 0.05M Tris-Buffer (pH=7.5).The tissue was then again rinsed with NaCl solution. The samples wereultracentrifuged and treated with pepsin and HCl. The amount of pepsinand HCl was determined using the wet weight of the sample. The remainingsample was tested.

Sample 5: The tissue was rinsed NaCl solution followed by ultrapurewater. The sample was then treated with sterile RNase A (100 μg/mL) andDNase I (150 IU/mL) with 10 mmolMgCl2, in 0.05M Tris-Buffer (pH=7.5) andthen washed with citrate buffer with a pH=4.0 for 72 hours. The sampleswere ultracentrifuged and treated with pepsin and HCl. The amount ofpepsin and HCl was determined using the wet weight of the sample. Theremaining sample was tested.

Sample 6: The tissue was rinsed with NaCl solution followed by ultrapurewater. The sample was then treated with sterile RNase A (100 μg/mL) andDNase I (150 IU/mL) with 10 mmolMgCl2, in 0.05M Tris-Buffer (pH=7.5).The samples were ultracentrifuged and treated with pepsin and HCl. Theamount of pepsin and HCl was determined using the wet weight of thesample. The remaining sample was tested.

Sample 7: The tissue was rinsed with ultrapure water. The sample wasthen treated with sterile RNase A (100 μg/mL) and DNase I (150 IU/mL)with 10 mmolMgCl2, in 0.05M Tris-Buffer (pH=7.5) and then washed withcitrate buffer with a pH=4.0 for 72 hours. The tissue was then againrinsed with NaCl solution. The samples were ultracentrifuged and treatedwith pepsin and HCl. The amount of pepsin and HCl was determined usingthe wet weight of the sample. The remaining sample was tested.

Sample 8: The tissue was rinsed with ultrapure water. The sample wasthen treated with sterile RNase A (100 μg/mL) and DNase I (150 IU/mL)with 10 mmolMgCl2, in 0.05M Tris-Buffer (pH=7.5). The tissue was thenagain rinsed with NaCl solution. The samples were ultracentrifuged andtreated with pepsin and HCl. The amount of pepsin and HCl was determinedusing the wet weight of the sample. The remaining sample was tested.

Sample 9: The tissue was rinsed with ultrapure water. The sample wasthen treated with sterile RNase A (100 μg/mL) and DNase I (150 IU/mL)with 10 mmolMgCl2, in 0.05M Tris-Buffer (pH=7.5) and then washed withcitrate buffer with a pH=4.0 for 72 hours. The samples wereultracentrifuged and treated with pepsin and HCl. The amount of pepsinand HCl was determined using the wet weight of the sample. The remainingsample was tested.

Sample 10: The tissue was rinsed with ultrapure water. The sample wasthen treated with sterile RNase A (100 μg/mL) and DNase I (150 IU/mL)with 10 mmolMgCl2, in 0.05M Tris-Buffer (pH=7.5). The samples wereultracentrifuged and treated with pepsin and HCl. The amount of pepsinand HCl was determined using the wet weight of the sample. The remainingsample was tested. Sample 11: The sample was treated with sterile RNaseA (100 μg/mL) and DNase I (150 IU/mL) with 10 mmolMgCl2, in 0.05MTris-Buffer (pH=7.5) and then washed with citrate buffer with a pH=4.0for 72 hours. The tissue was then again rinsed with NaCl solution. Thesamples were ultracentrifuged and treated with pepsin and HCl. Theamount of pepsin and HCl was determined using the wet weight of thesample. The remaining sample was tested. Sample 12: The sample wastreated with sterile RNase A (100 μg/mL) and DNase I (150 IU/mL) with 10mmolMgCl2, in 0.05M Tris-Buffer (pH=7.5). The tissue was then againrinsed with NaCl solution. The samples were ultracentrifuged and treatedwith pepsin and HCl. The amount of pepsin and HCl was determined usingthe wet weight of the sample. The remaining sample was tested.

Sample 13: The sample was treated with sterile RNase A (100 μg/mL) andDNase I (150 IU/mL) with 10 mmolMgCl2, in 0.05M Tris-Buffer (pH=7.5) andthen washed with citrate buffer with a pH=4.0 for 72 hours. The sampleswere ultracentrifuged and treated with pepsin and HCl. The amount ofpepsin and HCl was determined using the wet weight of the sample. Theremaining sample was tested.

Sample 14: The sample was treated with sterile RNase A (100 μg/mL) andDNase I (150 IU/mL) with 10 mmolMgCl2, in 0.05M Tris-Buffer (pH=7.5).The samples were ultracentrifuged and treated with pepsin and HCl. Theamount of pepsin and HCl was determined using the wet weight of thesample. The remaining sample was tested.

Sample 15: The tissue was rinsed with NaCl solution followed byultrapure water. The sample was then treated with TritonX-Sodiumdeoxycholate-PBS solution at 0 to 25 degrees C. for at least 24 hours.The tissue was rinsed and then treated with sterile RNase A (100 μg/mL)and DNase I (150 IU/mL) with 10 mmolMgCl2, in 0.05M Tris-Buffer (pH=7.5)and then washed with citrate buffer with a pH=4.0 for 72 hours. Thesamples were ultracentrifuged and treated with pepsin and HCl. Theamount of pepsin and HCl was determined using the wet weight of thesample. The remaining sample was tested.

Sample 16: The tissue was rinsed with NaCl solution followed byultrapure water. The sample was then treated with TritonX-Sodiumdeoxycholate-PBS solution at 0 to 25 degrees C. for at least 24 hours.The tissue was rinsed and then treated with sterile RNase A (100 μg/mL)and DNase I (150 IU/mL) with 10 mmolMgCl2, in 0.05M Tris-Buffer(pH=7.5). The samples were ultracentrifuged and treated with pepsin andHCl. The amount of pepsin and HCl was determined using the wet weight ofthe sample. The remaining sample was tested.

DNA contents of the treated samples as described above were determined.As shown in FIG. 2, the y axis is ng of DNA per gram of collagenformulation, and the x axis is the sample designation as noted above.These data show the relative efficacy of the citrate rinse (samples 4,6, 11) and the DNAse/RNAse steps (samples 3, 4, 5, 6, 7, 8). Triton Xalso had some efficacy (Samples 15 and 16). For reference, the DNAcontent of the native tissue is approximately 500,000 to 3,000,000 ngDNA/g tissue.

The treated samples were compared with those in native tissues and incommercially available scaffolds, using the processing techniquesdescribed above. The collagen scaffold described herein was testedagainst: 1) native tissue, 2) Surgifoam, 3) TissueMend. Collagen content(FIG. 3, panel A), GAG content (FIG. 3, panel B), and phophatidylcholinecontent (FIG. 3, panel C) as a measure of retained cellular membranewere investigated. The results are listed below.

DNA content (ng DNA/g tissue or scaffold) for treated and untreatedscaffolds was compared with that in native tissue (no treatment),TissueMend and Surgifoam (two FDA approved scaffolds). The treatment ofthe scaffold with techniques to remove DNAs as described above reducedthe DNA content in the scaffold to less than 20% of that found in thenative tissue. FIG. 3, panel A.

GAG content (ug GAG/g tissue or scaffold) for treated and untreatedscaffolds was compared with that of native tissue (no treatment),TissueMend and Surgifoam (two FDA approved scaffolds). The treatment ofthe scaffold with techniques described above to remove the GAG reducedthe GAG in the scaffold by over 30%. FIG. 3, Panel B.

Phospholipid content (uM/mg) of the native tissue, untreated scaffoldand treated scaffold was compared with that in Surgifoam and TissueMend(two FDA approved scaffolds). The treatment of the scaffold withtechniques described above to remove the phospholipid reduced thephospholipid in the scaffold to a level less than 20% that found in thenative tissue. FIG. 3, panel C.

Example 3 Chemical Neutralization of the Pepsin Content in a Scaffold

Scaffolds were made from extracellular matrix proteins using a pepsindigestion. After digestion, one group had no further treatment, whilethe second group was treated with a chemical to inactivate the pepsin.

Scaffolds were made from extracellular matrix proteins using a pepsindigestion. After digestion, one group had no further treatment, whilethe second group was treated with a strong base to inactivate thepepsin.

Briefly, a strong base (e.g., KOH or NaOH or LiOH) at a suitableconcentration was added into a collagen slurry in a dropwise manner tobring the pH value to above 4.0. Additional strong base was added tobring the pH of the slurry to 7.0 or greater. Once the slurry reachedits target pH range, the solution is kept there for a suitable period oftime, e.g., 1 to 10 minutes.

After inactivation of the pepsin, the pH of the slurry was returned to apH between 7.0 and 8.0 by the addition of a buffer with a pK of between7 and 8, such as a buffer containing TAPSO, HEPES, TES, MOPS, CacodylateSSC or Succinic acid.

As shown in FIG. 4, the level of active pepsin in a collagen materialreduced by around 80% as compared to the active pepsin level before theinactivation treatment.

Example 4 Testing Collagen Sponge with Calcium Added into CollagenSlurry

Methods: Five collagen sponges containing three different concentrationsof CaCl2 (high, medium and low), were prepared. The sponges wereprepared by taking collagen slurry, and adding a 30 mM, 60 mM or 90 mMsolution. The slurry was then lyophilized. The sponges were thenrehydrated with water to result in a specific collagen concentration.The collagen slurry was neutralized using a buffer with a pK of between7 and 8 (Cellgro, Mediatech, Inc, Herndon, Va.) and enough 7.5% sodiumbicarbonate (Cambrex BioScience Walkersville, Inc., Walkersville, Md.)to neutralize the acidic slurry to a pH of 7.4. Five 1.0 mL aliquots ofthe each concentration of neutral collagen gel was transferred intowells of a 24-wellplate and warmed until gelation occurred. The gelswere then lyophilized to make the collagen-calcium sponges.

0.75 mL platelet-rich plasma (PRP) containing plasma, platelets and ananticoagulant was placed on two sponges of each type to see if a clotformed in the collagen-calcium sponge. Two sponges of each concentrationwere also compressed and then PRP containing an anticoagulant was addedto see if that affected clot formation.

Results: After 10 minutes, clotting did occur in the 90 mM CaCl₂solution sponge. The lower calcium solutions did not clot as well, butsome initial clotting did occur. It did not matter whether or not thesponge was compressed before PRP was added. The collagen sponges withcalcium allowed the clot to form within the sponge at all concentrationsof calcium, even when the blood components were drawn into a tubecontaining an anticoagulant. The collagen-calcium was able to causeclotting of the anticoagulated blood product in the absence of any addedthrombin.

Example 5 Testing HDBC Sponge with Calcium and a Second Lyophilization

Method: To make the collagen sponges, acid soluble collagen slurry waslyophilized, and rehydrated with water to have a collagenconcentration>10 mg/ml. The collagen slurry was neutralized using HEPESBuffer (Cellgro, Mediatech, Inc, Herndon, Va.) and sodium bicarbonate(Cambrex BioScience Walkersville, Inc., Walkersville, Md.) to neutralizethe acidic slurry to a pH of 7.4. The pH neutral collagen gel wasincubated at 37 degrees C. to allow for gelation of the collagenhydrogel and self-assembly of the collagen fibers, and the gel was thenlyophilized to make a collagen sponge. The collagen sponge was cut intothirds and each third was placed into its own Petri dish. One sponge wascovered with a low concentration solution of calcium, one sponge wascovered with a medium concentration of calcium, and one sponge wascovered with a high concentration of calcium. These three sponges wereplaced in new Petri dishes and were then placed back into thelyophilizer for three days.

The collagen sponges were removed from the lyophilizer and 0.75 mL of asolution containing an anticoagulant, platelet, plasma and white bloodcells was placed on each sponge. After 10 minutes, the sponges were putinto a 50 cc test tube, and shaken and vortexed to test their structuralrigidity.

Results: It was found that a clot formed in each of the sponges within10 minutes of the blood components being added. The collagen spongeswith calcium allowed the clot to form within the sponge at allconcentrations of calcium, even when the blood components were drawninto a tube containing an anticoagulant. The collagen-calcium was ableto cause clotting of the anticoagulated blood product in the absence ofany added thrombin.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific embodiments of the invention described herein. Suchequivalents are intended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety.

I claim:
 1. A collagen scaffold comprising: a self-assembly of collagenfibers formed by heating a neutralized collagen slurry; wherein theneutralized collagen slurry has a collagen concentration of at least 10mg/ml; and wherein the collagen scaffold is compressible.
 2. Thecollagen scaffold of claim 1, wherein the collagen scaffold isexpandable.
 3. The collagen scaffold of claim 1, further comprisingcalcium.
 4. The collagen scaffold of claim 1, wherein the collagenfibers comprise calcium.
 5. The collagen scaffold of claim 1, whereinthe collagen slurry comprises calcium.
 6. The collagen scaffold of claim1, wherein the collagen scaffold is a sponge.
 7. The collagen scaffoldof claim 1, wherein the collagen scaffold is capable of absorbing atleast 3 ml of blood.
 8. The collagen scaffold of claim 1, wherein thecollagen scaffold is capable of absorbing at least 9 ml of blood.
 9. Thecollagen scaffold of claim 1, wherein the collagen scaffold iscylindrical.
 10. The collagen scaffold of claim 9, wherein the collagenscaffold is about 22 mm in diameter.
 11. The collagen scaffold of claim9, wherein the collagen scaffold is about 30 mm in length.
 12. Thecollagen scaffold of claim 1, wherein the collagen scaffold comprisescollagen obtained from a bovine knee.
 13. The collagen scaffold of claim1, wherein the collagen scaffold comprises atelocollagen.
 14. Thecollagen scaffold of claim 1, comprising less than about 25 ug/g ofnucleic acids.
 15. The collagen scaffold of claim 1, comprising lessthan about 50,000 ng/g of DNA.
 16. The collagen scaffold of claim 1,comprising less than about 5,000 uM/g of phospholipids.
 17. The collagenscaffold of claim 1, comprising less than about 2,000 ug/g of GAG. 18.The collagen scaffold of claim 1, further comprising a therapeuticprotein.
 19. A collagen scaffold, comprising: a self-assembly ofcollagen fibers, calcium, less than about 50,000 ng/g of DNA, less thanabout 5,000 uM/g of phospholipids, and less than about 2,000 ug/g ofGAG, wherein the collagen scaffold is compressible, and wherein thecollagen scaffold is capable of absorbing at least 3 ml of blood. 20.The collagen scaffold of claim 19, wherein the collagen scaffold isexpandable.
 21. The collagen scaffold of claim 19, further comprisingcalcium.
 22. The collagen scaffold of claim 19, wherein the collagenfibers comprise calcium.
 23. The collagen scaffold of claim 19, whereinthe collagen scaffold comprises collagen obtained from a bovine knee.24. The collagen scaffold of claim 19, wherein the collagen scaffoldcomprises atelocollagen.
 25. The collagen scaffold of claim 19,comprising less than about 25 ug/g of nucleic acids.
 26. A collagenscaffold prepared by a process comprising the steps of: preparing aneutralized collagen slurry by mixing a collagen slurry with a bufferand then with a solution containing calcium, wherein the neutralizedcollagen slurry has a collagen concentration of at least 10 mg/ml;exposing the neutralized collagen slurry to a temperature sufficient tocause self-assembly of collagen fibers within the neutralized collagenslurry; and freeze drying the neutralized collagen slurry to a producethe collagen scaffold that is compressible and expandable.
 27. Thecollagen scaffold of claim 26, wherein freeze drying the heatedneutralized collagen slurry to produce the collagen scaffold compriseslyophilizing the neutralized collagen slurry.
 28. The collagen scaffoldof claim 26, further comprising the step of adjusting the pH of theneutralized collagen slurry to between 6.2 and 7.6.
 29. The collagenscaffold of claim 26, wherein heating the neutralized collagen slurryforms the collagen scaffold comprised of self-assembled collagen fibers.